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Pharmacological Research 99 (2015)370376
Contents lists available at ScienceDirect
Pharmacological Research
journal homepage: www.elsevier .com/ locate /yphrs
Aiming for allosterism: Evaluation ofallosteric modulators ofCB1in aneuronal model
Alex Straiker,Jos Mitjavila, Danielle Yin, Anne Gibson, Ken Mackie
Department of Psychological and Brain Sciences,Gill Center for Biomolecular Science, Indiana University, 1101 E, 10th St,Bloomington, IN 47405,
United States
a r t i c l e i n f o
Article history:
Received 10 June 2015Received in revised form 16 July 2015
Accepted 17 July 2015
Available online 23 July 2015
Keywords:
Allosterism
Allosteric
Orthosteric
Cannabinoid
Depolarization-induced suppressionof
excitation
Tetrahydrocannabinol
Excitatorypostsynaptic current
a b s t r a c t
Cannabinoid pharmacology has proven nettlesome with issues ofpromiscuity a common theme among
both agonists and antagonists. One recourse is to develop allosteric ligands to modulate cannabinoid
receptor signaling. Cannabinoids have come late to the allosteric table. The first-generation negative
and positive allosteric modulators (NAMs and PAMs) represent an important first effort. However, most
studies have relied on synthetic agonists, often tested in over-expression systems rather than a defined
neuronal model system that utilizes endogenously synthesized and released cannabinoids.
We have systematically examined first-generation NAMs and a PAM on endocannabinoid modula-
tion of synaptic transmission in cultured autaptic hippocampal neurons. These neurons exhibit CB1and 2-arachidonoyl glycerol (2-AG)-mediated depolarization induced suppression of excitation (DSE)
and therefore serve as a model to test CB1 modulators in a neuronal model ofendogenous cannabinoid
signaling.
WefindORG27569,PSNCBAM-1,andPEPCAN12attenuateDSEanddonotdirectly inhibitCB1 receptors.
Ofthese PSNCBAM-1 is themost efficacious while PEPCAN12 has the distinction ofbeing an endogenous
NAM. The reported NAMs pregnenolone and hemopressin as well as the reported PAM lipoxin A4 are
without effect in this model ofendocannabinoid signaling.
In summary, three ofthe allostericmodulators evaluated function in amannerconsistentwith alloster-
ism in a neuronal 2-AG-based model ofendogenous cannabinoid signaling. 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Cannabinoid receptors were first identified in the early 1990s
[1,2]. These G protein-coupled receptors mediate most of the
salient effects of marijuana consumption and also are also key
components of an endogenous signaling system that is both phy-
logenetically highly conserved[3] and active throughout thebody.
Thecannabinoid research field benefited from the early identifica-
tionof potent andefficacious orthosteric agonists(i.e.WIN55212-2
and CP55940) and antagonists (i.e. SR141716, AM251), all still in
experimental use. However, due to the sometimes unfavorable
therapeutic profiles of the orthosteric CB1 ligands, there has been
a strong interest in the development of allosteric modulators at
Abbreviations: DSE, depolarization-induced suppressionof excitation; 2-AG,2-
arachidonoly glycerol;NAM, negative allostericmodulator; PAM,positive allosteric
modulator; 9THC, tetrahydrocannabinol; EPSC, excitatory postsynaptic current;
pERK,phospho extracellular signaling related kinase. Corresponding author.
E-mail address: [email protected] (A. Straiker)
CB1. Negativeallostericmodulators (NAMs) inhibit binding and/or
signaling while positive allosteric modulators (PAMs) potentiate
bindingand/or signaling by an orthosteric agonist [4]. NAMs/PAMs
may act in a variety of ways but in its simplest form the concept
is that a receptormay have one ormore additional allosteric sites
that when engaged will modulate orthosteric signaling. This usu-
ally occursby altering thebinding kineticsof the orthostericligand
and/or by potentiating/inhibiting the receptors signaling via one
or more signaling pathways. A major advantage of allosteric mod-
ulators is the potential to modulate only activated receptors. This
selectivity is especially relevant to the nearly ubiquitous cannabi-
noidsignaling systemwithits highlevelofendogenousactivityand
consequent risk of off-target action during a therapeutic interven-
tion.
Allosteric modulation is not a new idea. For instance several
important classes of drugs are allosteric modulators at GABAAreceptors (e.g. benzodiazepines [5]). However the first two CB1NAMs,ORG27569 andPSNCBAM-1werecharacterized lessthan10
years ago [69], while the first CB1 PAM has been described more
recently [10]. These first generation allosteric modulators gener-
atedexcitementbut additionalstudieshave foundthattheypossess
http://dx.doi.org/10.1016/j.phrs.2015.07.017
1043-6618/ 2015 Elsevier Ltd. All rights reserved.
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A. Straiker et al. / Pharmacological Research 99 (2015) 370376 371
complex pharmacological profiles. For instance,ORG27569 lowers
cAMP inhibition consistent with NAM activity [6]. But ORG27569
also increases binding of CP55940 [9], stimulates CB1 internal-
ization [6], and potentiates G-mediated pERK production [11].Similarly PSNCBAM-1 has been demonstrated to enhance binding
affinity [8]. Adding to thecomplexity, a recentstudythatexamined
ORG27569 and PSNCBAM-1 found that even the reported NAM-
like reduced CB1 internalization [11], may in fact be a secondary
consequence of enhanced orthosteric binding and consequent
desensitization [12]. Therefore these compounds do notbehave as
pure NAMs under the conditions that they have been tested thus
far.
PEPCAN12 is a member of a novel class of endogenous com-
pounds [13]. Discovered in2012, PEPCAN12may beanendogenous
CB1-modulating peptide acting as a NAM. More recently levels
of the important steroid hormone precursor, pregnenolone, were
reported to beupregulated in vivo by9THC; pregnenolone acted
as a NAM for 9THC at CB1 receptors [14]. While this is a highly
interestingfinding,the interactionofpregnenolonewithCB1 recep-
torsactivated byendogenous cannabinoidswasnotevaluated.And
lastly lipoxin A4was reportedas one of the firstCB1PAMs [10]. The
identificationof aPAMis ofparticular interestchieflybecausemany
of thetherapeuticapplicationsforcannabinoidsinvolveCB1activa-
tion.PAMsofferthepossibilityof targetinga subsetof CB1 receptors(i.e. those currently active). Additionally, a pathway-specific PAM
may further limit off-target action and may avoid undesired psy-
choactiveeffects,particularlywhenCB1is activatedbyendogenous
ligands.
The pharmacology of allosterism can become quite com-
plex, particularly because allosteric modulation can be probe-
dependent, i.e. it may depend greatly on the orthosteric ligand
being used [4]. Generally speaking, the therapeutic use of an
allosteric modulator will involve the endogenous ligand, yet the
characterizations of most CB1 allosteric modulators have used
synthetic agonists such as CP55940 or WIN55212-2 or the phy-
tocannabinoid, 9THC.Wehave examined the effect of a range of
first-generationCB1 allostericmodulatorson synaptictransmission
using autaptic hippocampal neurons. These cultures are a well-characterized model of endogenous cannabinoid signaling that
expresses themachinery to produce andmetabolize endocannabi-
noids aswell as presynaptic CB1receptors [1517]. Depolarization
of these neurons induces depolarization induced suppression of
excitation (DSE) a form of retrograde inhibition involving endo-
cannabinoids and CB1 receptors found in many brain regions [18].
Theautapticmodel is well-suited to an examination of allosterism
with the endocannabinoids since we have established that DSE is
mediatedby theendocannabinoid2-arachidonoylglycerol[15].We
now report our study of first-generation allosteric modulators in a
neuronal model of endogenous CB1signaling.
2. Methods
2.1. Hippocampal culture preparation
All procedures used in this study were approved by the Ani-
mal Care Committee of Indiana University and conform to the
Guidelines of the National Institutes of Health on the Care and
Use of Animals. Mouse (CD1 strain) hippocampal neurons iso-
lated from the CA1-CA3 region were cultured on microislands as
describedpreviously [19,20]. Neuronswereobtainedfromanimals
(agepostnatal day02)andplatedontoa feederlayerof hippocam-
pal astrocytes that had been laid down previously [21]. Cultures
were grown in high-glucose (20mM)DMEM containing 10%horse
serum, without mitotic inhibitors and used for recordings after 8
days in culture and for nomore than3h after removal fromculture
medium.
2.2. Electrophysiology
When a single neuron is grown on a small island of permis-
sive substrate, it forms synapsesor autapsesonto itself. All
experimentswere performedon isolated autaptic neurons.Whole
cell voltage-clamp recordings from autaptic neurons were carried
out at room temperature using an Axopatch 200A amplifier (Axon
Instruments, Burlingame, CA).Theextracellular solutioncontained
(in mM) 119 NaCl, 5 KCl, 2.5 CaCl2, 1.5 MgCl2, 30 glucose, and
20 HEPES. Continuous flow of solution through the bath chamber
(2ml/min) ensured rapid drug application and clearance. Drugs
were typically prepared as stocks, and then diluted into extracel-
lular solution at their final concentration and used on the same
day.
Recording pipettes of 1.83Mwere filled with (in mM) 121.5
KGluconate,17.5KCl,9NaCl,1 MgCl2, 10HEPES,0.2EGTA, 2MgATP,
and 0.5 LiGTP. Access resistance and holding current were moni-
tored andonly cellswith both stable access resistance and holding
currentwereincludedfor dataanalysis. Conventionalstimuluspro-
tocol: the membrane potentialwas held at70mV and excitatory
postsynapticcurrents(EPSCs)were evokedevery20s by triggeringan unclamped action current with a 1.0ms depolarizing step. The
resultant evoked waveform consisted of a brief stimulus artifact
anda largedownwardspike representing inward sodiumcurrents,
followedby the slowerEPSC. Thesizeof therecordedEPSCswascal-
culatedbyintegratingthe evokedcurrentto yielda charge value(in
pC). Calculating the charge value in thismanner yields an indirect
measure of the amount of neurotransmitter released while mini-
mizing theeffectsof cabledistortiononcurrentsgenerated far from
the site of the recording electrode (the soma). Data were acquired
at a sampling rate of 5kHz.
DSE stimuli: After establishing a 1020s 0.5Hz baseline, DSE
was evoked by depolarizing to 0mV for 50ms, 100ms, 300ms,
500ms, 1s, 3s and 10s, followed in each case by resumption of
a 0.5Hz stimulus protocol for 2080+ seconds, allowing EPSCs torecover to baseline values. This approach allowed us to determine
the sensitivity of the synapses to DSE induction. To allow compar-
ison, baseline values (prior to the DSE stimulus) are normalized
to one. DSE inhibition values are presented as fractions of 1, i.e. a
50% inhibition from the baseline response is 0.50 standard error
of the mean. Thex axis of DSE depolarizationresponse curves are
log-scalesecondsof thedurationof thedepolarizationused toelicit
DSE.
Depolarization response curves are obtained to determine
pharmacologicalpropertiesofendogenous2-AGsignalingbydepo-
larizingneurons forprogressively longerdurations(50ms, 100ms,
300ms, 500ms, 1s, 3s and 10s). The data are fitted with a nonlin-
ear regression, allowing calculation of an ED50, the effective dose
or durationof depolarization atwhicha 50% inhibition is achieved.Statistical significance in these curves is taken as 95% confidence
interval.
2.3. Drugs
Concentrations of the drugs were tested at 1M consideredto be a concentration that was high enough to expect to see an
effectbutnotso high as to introducean artifact or off-target effect.
The exception was PEPCAN12 which has been found to be effec-
tive at very low concentrations. Thus we tested this compound at
the lower concentration of 200nM. PEPCAN12 was the generous
gift ofDr. JuergGertsch (Universitt Bern). ORG27569was the gen-
erous gift of Dr. Ruth Ross (University of Toronto). Hemopressin
was the generous gift of Dr. Lakshmi Devi (Mt. Sinai University).
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372 A. Straiker et al. / Pharmacological Research 99 (2015) 370376
PSNCBAM-1, lipoxin A4 and pregnenolone were purchased from
TocrisBioscience (Bristol, UK), CaymanChemicals (AnnArbor,MI),
and Sigma Aldrich (St. Louis,MO), respectively.
3. Results
We examined the signaling properties of candidate CB1allosteric modulators in autaptic hippocampal neurons, a model
of endogenous cannabinoid signaling. CB1activation inhibits neu-rotransmitter release in these neurons via subunit-dependentinhibition of calcium channels, resulting in a smaller excitatory
postsynaptic current (EPSC) [15,22,23]. A negative allosteric mod-
ulator (NAM) at CB1 should not inhibit EPSCs on its own but it
should reduce the inhibition induced by CB1 activation. Because
2-AG mediated depolarization induced suppression of excitation
(DSE) can be elicited in these neurons, we can test the effect of a
given candidate NAM on endogenous 2-AG signaling by depolar-
izing neurons for progressively longer durations (50ms, 100ms,
300ms, 500ms, 1 s, 3 s and 10s). A NAM should shift the DSE
response curve up and/or to the right, while a PAM should shift
the curve down and/or to the left.
3.1. ORG27569 and PSNCBAM-1 act in a manner consistent with
negative allostericmodulation
We first tested the twooriginal negative allosteric modulators:
ORG27569 [6,7,9] and PSNCBAM-1 [8]. ORG27569 did not directly
inhibit EPSCs directly at 1M (Fig. 1C, relative EPSC charge (1MORG): 0.990.01, n=6). We also found that at 1M ORG27569did not shift the ED50 of the DSE depolarization response curve
(Fig. 1A, ED50(baseline): 1.25(1.151.36) ED50(ORG27569):1.30s
(1.121.51); n=5, overlapping 95% CIs). However, there was a
statistically significant difference in inhibition for longer depo-
larizations (Fig. 1A, relative EPSC charge (baseline, 10s depol):
0.380.04; (ORG): 0.590.03, n=6; p0.05 1 way ANOVA with Bonferroni posthoc
test).
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A. Straiker et al. / Pharmacological Research 99 (2015) 370376 373
Fig. 2. PSNCBAM-1 substantially inhibits DSE. (A) Depolarization response curve
before (blacksquares)and after/during 1MPSNCBAM-1treatment(red triangles).
(B) Averaged DSE (3s depolarization)time courses beforeand after drug. (C) Effect
of1MPSNCBAM-1aloneon EPSCs inWTneurons. (For interpretation ofreference
to colorin this figurelegend,the readeris referredto thewebversionof this article.)
3.3. Pregnenolone does not attenuate 2-AG signaling in autaptic
neurons
Lastly among the NAMswe tested the steroid hormone precur-
sor,pregnenolone. Ina recent report pregnenolonewas foundto actin a manner consistent with negative allosteric modulation of CB1[14] a potentially significant finding for this important hormone
precursor. However, we found that pregnenolone at 1M waswithout effect on endocannabinoid signaling in autaptic neurons
(Fig.5, ED50(pre): 2.7s (2.43.0); ED50(Preg1 M): 2.2s (2.02.4);Relative EPSC charge after preg: 1.010.01, n=5).
3.4. Lipoxin A4 antagonizes cannabinoid inhibition of
neurotransmission
To finish on a positive note, we turned to the putative PAM
lipoxin A4 [10]. In contrast to NAMs, wewould expect lipoxin A4
to shift the DSE curve to the left. However we found that if any-
thing 1M lipoxin A4 shifted the curve to the right, suggesting
Fig. 3. PEPCAN12 inhibits DSE. (A) Depolarization response curve before (black
squares)and after/during200nM PEPCAN12treatment(red triangles). (B)Averaged
DSE(series of depolarizations 100ms to 1s) timecourses beforeand after drug.(C)
Effectof 200nM PEPCAN12aloneon EPSCs.(For interpretation of referenceto color
in this figure legend, thereader is referred to theweb version of this article.)
antagonismofCB1signaling (Fig. 6, ED50(baseline): 2.2s (2.12.4);
(drug): 3.3 s (3.03.6); n=5; non-overlapping 95% CIs). Because
lipoxin A4 did not inhibit EPSCs when applied on its own (Fig. 6C,relative EPSC charge: 1.020.02, n=5), it is possible that lipoxin
A4 serves as a NAM rather than a PAMwith2-AG as the ligand.
4. Discussion
Allostericmodulators of cannabinoid receptor signaling are rel-
atively new arrivals on the pharmacotherapeutic stage. Since CB1receptors (and the associated therapeutic potential) are abundant
throughout the CNS and since 2-AG is implicated in many of the
known forms of CB1-mediated plasticity, we have tested several
of the first-generation negative andpositive allosteric modulators
in a neuronal model that uses 2-AG as the endocannabinoid in an
endogenous retrograde signaling system. Our chief finding is that
two of the six candidates act in a manner consistent with their
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Fig. 4. Hemopressin does not inhibit neurotransmission via CB1. (A) DSE response
beforeandafter1Mhemopressin. (B)Effectof 2Mhemopressin (pipette)on DSE
at indicated time alone on EPSCs inWT neurons.
Fig.5. Pregnenolonedoesnot inhibit neurotransmissionvia CB1. (A)Depolarization
response curve before (black squares) and after/during 1M pregnenolone treat-ment (red triangles).(B) Effectof 1Mpregnenolonealone onEPSCs inWTneurons.
(For interpretation of reference to color in this figure legend, the reader is referred
to theweb version of this article.)
predicted allosteric modulation while the remaining compounds
have a diversity of actions, includingagonist effects.
While our experiments can rule out an allosteric candidate
(in regard to 2-AG modulation of synaptic transmission), they
cannot rule one in: the definitive identification of a compound
as an allosteric agent requires binding studies that character-
ize the impact of that compound on the binding kinetics of an
orthosteric ligand.We donot thereforeconcludethat a given com-
pound is or is not an allosteric modulator but have determined
whether a compound does/does not act in a manner consis-
tent with allosteric modulation in the specific pathway of 2-AG
inhibition of glutamatergic transmission. And importantly all is
not lost for compounds that fail to act as negative allosteric
Fig. 6. ThePAM LipoxinA4 (LPA4)inhibitsDSE in autaptic neurons. (A)Depolariza-
tion response curve before (black squares) and after/during 1M LPA4 treatment
(red triangles). (B) Averaged DSE (3s depolarization) time courses before and after
drug. (C) Effect of 1M LPA4 alone on EPSCs inWT neurons. (For interpretation of
reference to color in this figure legend, the reader is referred to the web version of
this article.)
modulators in a neuronalmodel of inhibition of synaptic transmis-
sion. As has been shown for ORG27569, a given compound may
serve as a negative allosteric modulator for one signaling path-
way but not another. Indeed thismay be a preferredoutcome. The
CB1antagonist SR141716(akaRimonabant/Accomplia) ranafoulofantihedonic properties during clinical use followingEMAapproval
[25,26] that led to its failure to be approved in the US and even-
tual withdrawal from the market in Europe. A NAM that avoids
inhibition of thereceptors/pathwaysresponsible for thisantihedo-
nic actionmayproduce the desired therapeutic/cosmetic outcome
free of this side-effect. Conversely one of the allures of positive
allosteric modulators would be the ability to induce a therapeutic
effect without the undesired psychoactive responses, perhaps via
signalingpathway-specific actions.
Ourresultsadda notch infavorof ORG27569asaNAM.Asnoted
above ORG27569 has a mixed profile that includes an enhance-
ment of CP55940 binding as well as an unexpected enhancement
of pERK signaling [11]. Most studies of ORG27569 action have
examined its interaction with the synthetic CB1 agonist CP55940
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